Guinea Pig Model For Vaccine Potency Evaluation Against Vaccine BoHV-1 GG~-/tk~- And Immunological Characteristics Of The Mutant BoHV-1 GN~-/tk~-/gG~-

Infectious bovine rhinotracheitis?IBR?is a feverish and contagious infectious disease of cattle caused by Infectious bovine rhinotracheitis virus?IBRV?,also know as Bovine herpes virus type 1.It cause rhinotracheitis,vaginitis,conjunctivitis,enteritis and abortion.Since it lacks effective therapeutic drugs,control of the virus in many countries is achieved primarily through vaccination.Gene engineering vaccines are safe and have an excellent immunization ffficacy and won't recovery to ancestral virulence,most important,it could be differentiated from that induced by wild-type BoHV-1 by the use of a gene specific ELISA kit.The aim of the present study is using Guinea pig model to evaluate the efficacy of the double gene mutant BoHV-1 gG^-/tk^-marker vaccine and study the biological characteristics and immune efficiency of the triple gene mutant BoHV-1gN^-/tk^-/gG^-.The main contents are as follows:1.Establishment of guinea pig model for evaluation of BoHV-1 gG-/tk-double gene deletion vaccine?1?The infection model was established by direct challenge of wt BoHV-1 in guinea pigs,the main indicators include clinical symptoms and body temperature,nasal shedding of virus,pathological changes of tissues and detection of viral nucleic acid in the tissue.The results showed that the wild strain could induce characteristic clinical symptoms,nasal shedding of virus and pathological changes of tissues in guinea pigs,indicating the successful establishment of the infection model.?1?We evaluated the safety of vaccine BoHV-1 gG^-/tk^-.The main contents including clinical symptoms and body temperature monitoring,specific antibody gB against BoHV-1,neutralizing antibody and cytokines.The results showed that both inoculation routes with 10⁶TCID₅₀?10⁷TCID₅₀?10⁸TCID₅₀0 doses of gene deleted vaccine BoHV-1gG^-/tk^-can induce immune response in guinea pigs,no significant change in body temperature,no virus was isolated from the nasal swabs,clinical symptoms of each vaccination group were slight,indicating that the vaccine is safy and have a good immunogenicity.In addition,dose-effect relationship exists,furthermore,secondary immunization can effectively promote the production of antibodies and the immune responses induced by groups inoculated by intramuscular injection are stronger than that caused by intranasal inoculation.?3?Through immunized with the double gene mutant vaccine BoHV-1 gG^-/tk^-at10⁶TCID₅₀,10⁷TCID₅₀ and 10⁸TCID₅₀ dose by intramuscular immunization,then challenged with wt BoHV-1,evaluating clinical symptoms,body temperature,specific antibody gB against BoHV-1,neutralizing antibody and cytokines,we determined that the clinical symptoms of each group were shorter than that of the wt group,and there was no significant change in body temperature.No virus was isolated from the nasal swabs.And the antibody level indicated a dose-effect relationship.The results of lung lesion degree showed that the lesion degree was negatively correlated with the vaccination dose,and the higher the vaccination dose was,the less severe the lesion was after challenge.By comparing groups 10⁷TCID₅₀ and 10⁸TCID₅₀,there was no significant difference between the levels of gB antibody and cytokine between the two groups?p>0.05?,and the level of neutralizing antibody was only significantly different at 49d?p<0.05?.Moreover,we evaluated the immune response elicited in guinea pigs by the vaccine formulated with adjuvant,and compared with the group of same vaccine dose without adjuvant.The final result shows that when vaccine in combination with adjuvant induced stronger immune response,the level of specific gB and neutralizing antibody were higher than the same vaccine doses of intramuscular injection group,especially the neutralizing antibody.In addition,the adjuvant also promoted IFN-gamma production.2.Biological and immunological characteristics of the triple gene mutant BoHV-1gN^-/tk^-/gG^-This study includes the study of biological characteristics and the immune efficiency of the triple gene mutant BoHV-1 gN^-/tk^-/gG^-in rabbit.The first part mainly includes salvation of the recombinant virus,and the comparison of plaques size and growth curve with wild strains.The results showed that we successfully rescued the recombinant virus,compared with wild strain,the recombinant virus have smaller plaques,and its virus titer are lower than the wild strain,but the difference is not significant.The second part includes clinical symptoms and body temperature changes,nasal shedding of virus,antibody level detection,cytokine detection,sIgA antibody detection in nasal swabs,pathological changes of tissues and detection of viral nucleic acid in the tissue after inoculation and challenge.After inoculation,rabbits in each group showed typical clinical symptoms,and virus shedding also could be detected.Compared with the wt group,the duration of clinical symptoms and virus shedding were shorter.After inoculation,both intramuscular group and wt group are gB positive at 7 d,but the former rising slowly than the latter,however,intranasal inoculation group retain negative before challenge,and its the antibody level is far lower than the intramuscular injection group after challenge.Compared to other groups,intramuscular injection group induced neutralizing antibody more quickly,which can be detected at 7d,but rising slower than wt group.Intranasal inoculation group has not been detected after inoculation neutralizing antibodies,increased after the attack poison,but significantly lower than intramuscular injection group?p<0.001?.SIgA test results showed that sIgA concentration was the highest in the wild poison group.SIgA concentration in the intramuscular injection group was higher than that in the Intranasal inoculation group at 7d,and lower than the latter at all subsequent time points,but the difference was not significant?p>0.05?.